The enteric nervous system (ENS) is remarkable for its phenotypic diversity and regulation of complex enteric behaviors independently of CNS input. Research into the pathogenesis of the congenital aganglionosis of Hirschsprung's disease (HSCR) has provided critical information about genes and molecules that act early in development to regulate colonization of the bowel by enteric neural crest-derived cells (ENCDC) and neurogenesis. Less is known about the later acting genes or molecules, which contribute to enteric neuronal phenotypic diversity. The current proposal is designed to develop an understanding of mechanisms that underlie the acquisition of this diversity, which when abnormal, disturb GI motility without giving rise to HSCR. We propose to test the hypothesis that neurotransmitters of early-born neurons, which co-exist with still-plastic dividing precursors, influence the fates of uncommitted ENCDC and, consequently, the late-born neurons that develop from them. Altered activity of early developing neurons that affects subsequent neuronal development might account for the long-lasting abnormal motility that have been observed in animals after neonatal irritation of the gut or maternal separation, as well as reports in humans with functional GI disorders of a history of infection, memories of abdominal pain during childhood, or a history of stress and/or abuse. The ENCDC that give rise to serotonergic neurons, which are among the first to born in the ENS, are transiently catecholaminergic (TC) and express the norepinephrine transporter (NET). Genetic NET deletion (NETKO) interferes with development of enteric serotonergic neurons. Neuronal 5-HT biosynthesis depends on tryptophan hydroxylase 2 (TPH2) while that in enterochromaffin cells depends on TPH1. The ENS of mice lacking neuronal 5-HT (TPH2KO), but not that of mice lacking mucosal 5-HT (TPH1KO), is deficient in total and dopaminergic neurons, although brain dopamine is normal. 5-HT thus promotes enteric neurogenesis/survival and the differentiation of enteric, but not CNS, dopaminergic neurons. In addition to 5-HT, the newly discovered, cerebral dopaminergic neurotrophic factor (CDNF) also contributes to ENS dopaminergic development. We will test hypotheses that NE (through NET) and 5-HT affect respectively, development of early-born and late-born enteric neurons. We propose that 5-HT is necessary for development of of late-born neurons, but does not, by itself, specify their phenotype, for example, to be dopaminergic. That requires additional factor(s), such as CDNF. Specifically, we will use transgenic mice and pharmacological tools to analyze the roles that norepinephrine and NET play in development/survival of enteric serotonergic neurons as well as the roles that enteric neuronal 5-HT and CDNF play in development/survival of dopaminergic and other late-born enteric neurons. We will also now analyze, in vivo and in vitro, the motility of mutant and wild-type gut to test the idea that the ENS defects that we identify in ! the mutant mice disturb intestinal tranit and motor patterns in adults despite the absence of aganglionosis.